Human papillomavirus entry: late trafficking and establishment of infection

跨高尔基体网络（trans-golgi network，TGN）是HPV感染细胞质转运的中间靶细胞器 传染性侵入以前已经确定，病毒基因组需要核膜破裂 进入细胞核。我们最近发现，病毒基因组在前期与TGN分离， 微管聚集在微管组织中心。在有丝分裂的后期（中期），病毒 基因组从有丝分裂纺锤体极移向染色体。我们的数据表明 有丝分裂过程中微管介导的从负端到正端定向运输的方向性。初步 抑制剂研究指出动力蛋白和Kif 11是参与转运的马达蛋白。方向性的改变， 目的1将进一步研究微管介导转运的马达蛋白的作用。而不是 病毒基因组在前期从内吞区室流出，但仍存在于膜结合区室中。 在整个有丝分裂运输过程中的囊泡隔室。病毒基因组从膜囊泡流出， 核膜重组这些是范式转变的发现，最近得到了Day的证实。 等使用电子显微镜。目前尚不清楚HPV如何从核运输囊泡中排出。我们 假设HPV利用细胞途径溶解最终进入细胞核的囊泡， 目的（例如病毒感染）或意外。事实上，初步的数据表明，特定的亚型， 磷脂酶C，其敲低在核递送后的阶段严重削弱HPV 16感染，可能是 参与溶解囊泡膜。将在目标2中使用击倒测试假设， 敲除方法结合差异染色技术和EM。从运输囊泡释放是 在PML蛋白募集之前。PML核小体（NB）的另一种成分Sp100的募集， 在进入的基因组周围的PML NB组装中特别延迟。在缺乏PML蛋白的细胞中，病毒 基因组丢失，因此转录失活。在目标2中，我们将测试我们的假设，即招聘 特异性PML亚型的存在是Sp100募集延迟的根本原因， NB重组对于传入基因组的转录调控是重要的。我们最近描述了 一种新的细胞培养模型，允许有效感染原代角质形成细胞，用于研究PML的作用 相关细胞培养模型中的NB组分。 总的来说，我们提出的研究将填补一个巨大的空白，在理解微管介导的运输期间， 晚期HPV细胞内运输，并将帮助我们解开PML NB在即刻早期事件中的作用 HPV的生命周期对于建立感染至关重要。此外，该提案有可能揭示 一种迄今未被认识的细胞途径，用于溶解最终进入细胞核的囊泡。虽然投机 在这个时候，这些观察可能会引发对囊泡向细胞核运送货物的可能作用的重新思考 在有丝分裂中的作用及其在核内稳态中的作用。

The trans-golgi network (TGN) serves as intermediate target organelle of the cytoplasmic transport during HPV infectious entry. It has previously been established that nuclear envelope breakdown is required for viral genome to enter the nucleus. We recently found that viral genome dissociates from TGN in prophase and associates with microtubules to accumulate at the microtubule-organizing center. At later stages of mitosis (metaphase), viral genome moves away from the mitotic spindle poles towards chromosomes. Our data point to a switch in the directionality of microtubule mediated transport from minus end- to plus end-directed during mitosis. Preliminary inhibitor studies point to dynein and Kif11 as motor proteins involved in the transport. Shift in directionality and the role of motor proteins for microtubule mediated transport will be further studied in Aim 1. Rather than egressing from the endocytic compartment in prophase, viral genome is still found in a membrane-bound vesicular compartment throughout mitotic transport. Viral genome egresses from membrane vesicles after nuclear envelope reformation. These are paradigm shifting findings, which have recently been confirmed by Day et al using electron microscopy. It is unclear, how HPV achieves egress from the nuclear transport vesicles. We hypothesized that HPV utilizes a cellular pathway to dissolve vesicles ending up in the nucleus, whether on purpose (e.g. viral infection) or accidentally. Indeed, preliminary data suggest that specific isoforms of phospholipase C, whose knockdown severely impairs HPV16 infection at a stage after nuclear delivery, may be involved in dissolving the vesicular membrane. The hypothesis will be tested in Aim 2 using knockdown and knockout approaches combined with differential staining techniques and EM. Release from transport vesicles is preceded by PML protein recruitment. Recruitment of another component of PML nuclear bodies (NB), Sp100, is specifically delayed in PML NB assembling around incoming genome. In cells deficient for PML protein, viral genome is lost and therefore transcriptionally inactive. In Aim 2, we will test our hypothesis that the recruitment of specific PML isoforms is the underlying reason for delayed recruitment of Sp100 and that HPV-induced PML NB reorganization is important for transcriptional regulation of the incoming genome. We have recently described a novel cell culture model that allows efficient infection of primary keratinocytes for investigating the role of PML NB components in a relevant cell culture model. Overall, our proposed studies will fill a huge gap in the understanding of microtubule mediated transport during late HPV intracellular trafficking and will help us unravel the role of PML NB during the immediate early events of the HPV life cycle essential for establishing infection. Furthermore, the proposal has the potential to uncover a hitherto unrecognized cellular pathway to dissolve vesicles ending up in the nucleus. Even though speculative at this time, the observations may trigger a rethinking of possible roles of vesicular cargo delivery to the nucleus during mitosis and its role in nucleus homeostasis.